Regulation of the extracellular matrix by miRNA: impact on cancer metastases

Regulation of gene expression is critical for all cellular processes, with dysregulation often resulting in development of disease. Gene expression can be regulated at the post-transcriptional level through the function of small non-coding RNAs known as microRNAs (miRNA). miRNAs are short single stranded RNAs that control mRNA stability or translational repression through base pairing with regions within the 3' untranslated region of the target mRNA. A number of miRNAs have been identified that control the expression of different components of the extracellular matrix (ECM). This regulation is critical for normal organ biogenesis and function, as alterations in ECM specific miRNA expression have been associated with development of metastatic cancer. Aberrant miRNA expression is often due to hypo- or hypermethylation of these genes, a frequent occurrence in cancer. These studies demonstrate the importance of tightly regulated ECM associated gene expression. Further analysis of cancer associated miRNA expression and methylation patterns through real-time PCR based methods will aid in development of new diagnostics and treatments.

Highlights

miRNA expression is altered in cancer, often through aberrant methylation

Altered expression of miRNAs that regulate extracellular matrix gene expression is associated with development of metastatic cancer

Novel approaches exploiting miRNA technology will aid in development of new treatments and diagnostic tools

miRNA originates from miRNA genes that are transcribed by RNA polymerase II or III, forming a 33 base pair hairpin structure. This primary microRNA (pri-miRNA) transcript is then bound by an RNAse enzyme complex formed by Drosha and DGCR8/Pasha, cleaving the 5' and 3' ends of the hairpin. The processed pre-miRNA is exported to the cytoplasm through Exportin-5 in complex with Ran-GTP. In the cytoplasm, the pre-miRNA is cleaved to its mature length by the DICER/TRBP complex, removing the hairpin loop. The functional strand is loaded with the Argonaut proteins on to the RISC silencing complex. This final complex regulates gene expression via one of two different mechanisms: translational repression or mRNA degradation through rapid deadenylation and decapping (see figure The canonical pathway of miRNA biogenesis) (1, 2).

The extracellular matrix (ECM) functions as a cellular scaffold composed of secreted proteins and polysaccharides. The components of the ECM function in structural support, cell communication, migration, adhesion and signaling. miRNAs regulating different components of the ECM have been identified and correlated with normal organogenesis, however altered miRNA expression has been associated with development of metastatic cancers. Identification of disease specific miRNA expression patterns has prognostic value in some cases (3).

Development of metastases is dependent in part on the interaction between the cancer cell and the ECM. Increased levels of matrix metalloproteases (MMP) encourage detachment of cancer cells from the basement membrane; decreased levels of MMP inhibitors (TIMP) similarly encourage cancer cell migration. Increased levels of collagen are correlated with cell migration and proliferation and have been shown to alter miRNA expression. Decreased levels of fibronectin have been correlated with increased metastatic potential (3–5). miRNAs that regulate MMPs, TIMPs, fibronectin, and collagen have altered expression in metastatic cancers and are useful diagnostic and prognostic indicators.

Back to top miRNAs regulate the expression of MMPs and tissue inhibitors of MMPs

MMPs degrade ECM components during normal physiological processes such as embryonic development, tissue and bone remodeling and wound healing however, they are also thought to contribute to tumor invasion when left unchecked. miR-146b was identified as an inhibitor of MMP-16 in glioma cells and is associated with glioma invasion and migration. Knockdown of miR-146b promoted invasion in U373 cells while overexpression of the miRNA inhibited invasive behavior in vitro (6). Another miRNA, miR-21, was shown to target TIMP3, a tissue inhibitor of MMPs. Overexpression of TIMP3 has been associated with apoptosis and decreased cell invasion in smooth muscle cells and melanoma cell lines, suggesting a protective function (7, 8). TIMP3 inhibits MMP activity by binding to the active site of the protease. Elevated in glioma cells, miR-21 decreases TIMP3 availability, resulting in uninhibited MMP activity. Elevated miR-21 promotes the invasiveness of glioblastoma; inhibition of this miRNA leads to decreased migration and invasion (9). miR-221/222 has been shown to target TIMP3 in aggressive forms of non-small cell lung cancer and hepatocellular carcinoma (HCC) promoting HCC cell invasion and metastasis. Control of MMP activity through post transcriptional regulation of the proteases themselves or via regulation of inhibitors such as TIMP3, is critical for suppressing the invasive potential of cancer cells. Dysregulation of associated miRNAs are frequently indicative of aggressive forms of these cancers.

The collagen microenvironment regulates gene expression The let-7 family of miRNAs were originally characterized in C. elegans where they regulate key transitions in embryonic development (10). These RNAs were recently shown to be downregulated in human pancreatic ductal adenocarcinoma (PDAC) cells, a cancer that exhibits a collagen-rich fibrosis (11). This collagen environment has been associated with increased cell motility and increased MMP activity (12). Recent reports indicate that the collagen microenvironment itself may be repressing levels of let-7 miRNAs in PDAC cells in part through increased levels of MMP-14 (9). This observation illustrates the importance of the interplay between components of the ECM and involvement in cancer progression.

Back to top Repression of fibronectin expression is associated with metastatic hepatitis B virus (HBV) associated HCC

HBV associated HCC is an aggressive cancer with poor survival rates. The HBV X (HBX) protein is thought to play a role in the observed pathology of invasion and metastases (13). miR-143 was reported to be upregulated in p21-HBX transgenic mice and patients with metastatic HCC. Transcription of miR-143 is stimulated by nuclear factor kappa B (NFκB) and promotes invasion of HBV-HCC by repressing fibronectin type III domain containing 3b (FNDC3B) (5). FNDC3B is a member of the fibronectin family of ECM proteins that has been reported to be downregulated in tumor cells with increased metastatic potential.

Back to top Alterations in the miRNA methylome contribute to metastases

Epigenetic modification is a major mechanism for gene regulation. A number of tumor suppressors are frequently hypermethylated in different cancers. For example, hypermethylation induced silencing is the major mechanism for loss of E-cadherin in cancer. Cancer cell genomes also become globally hypomethylated, losing an estimated 20-60% of their 5-methylcytosine content, resulting in activation of previously repressed genes. Global hypomethylation not only affects protein-coding genes but also affects miRNA loci. Several studies have demonstrated altered miRNA profiles in cancer. For example, miR-148a, miR-34b/c, and miR-9 were found to be reactivated upon treating lymph node metastatic cancer cells with the demethylating agent 5-aza-2'-deoxycytidine. Reactivation of these miRNAs resulted in loss of motility, decreased cell growth, and inhibited metastasis formation in xenograft models. These miRNAs were also found to be hypermethylated in human malignancies and are associated with development of lymph node metastases, highlighting the role of epigenetic silencing of tumor suppressor miRNAs in metastatic cancer (14).

Regulation of ECM proteins by miRNA is often dysregulated in aggressive and metastatic forms of cancer. Increased levels of MMPs, collagens, and altered fibronectin expression is associated with increased cancer cell migration and invasion; hallmarks of metastases. Cancer specific miRNA signatures offer prognostic value as well as represent targets for drug development. miRNA mimics that increase expression of silenced miRNAs are being evaluated as a possible treatment strategy (15). For example, miR-122 is required for hepatitis C virus (HCV) genome accumulation in cultured liver cells. Delivery of a locked nucleic acid (LNA) modified oligonucliotide complementary to miR-122 was shown to decrease HCV viremia in primates with no evidence of side effects (15). This study provides evidence of the feasibility of delivering anti-miRNA oligonucleotides in vivo. Several types of antisense based miRNA inhibitors including LNA and various 2'-O-modifed oligonucleotides have been successfully used to silence miR-122 in mice; however effective systemic delivery remains a challenge. In addition, corrective overexpression of downregulated miRNAs may represent an additional treatment strategy with similar delivery challenges (16).

Hypermethylation of tumor suppressor miRNAs and hypomethylation of oncogene miRNAs represents a major mechanism for altered miRNA expression metastatic cancer. Further analysis of the methylation status of relevant miRNAs in different cancer states will aid in identification of biomarkers with diagnostic and prognostic value. In addition, treatment with hypomethylating agents such as 5-aza-2'-deoxycytidine has been shown to counteract hypermethylation of miR-127 in bladder tumors (17). These studies hold promise in bringing novel mechanisms for controlling cell migration and invasion from bench to bedside.